Surface-Modified Heavy Metal Nanoparticles, Compositions And Uses Thereof

ABSTRACT

Surface-modified heavy metal nanoparticles, including a heavy metal core and a coating layer, the coating layer having at least one ligand, conjugated to polyethylene glycol, the at least one ligand is selected from N-acetyl cysteine, albumin, cysteine, methionine, glutathione, amino thiols, thio-carboxylic acids, ammonia, amines, diamines or any combination thereof. Compositions including surface-modified heavy metal nanoparticles and uses thereof in treatment and diagnosis of various conditions.

FIELD OF THE INVENTION

The invention relates to the field of surface modified metalnanoparticles and their use in treatment and diagnosis of various healthrelated conditions.

BACKGROUND OF THE INVENTION

Nanoparticles are small object in a nano scale size, that behaves as awhole unit in terms of its transport and properties. Nanoparticles mayexhibit size-related properties that differ significantly from thoseobserved in fine particles or bulk materials. Methods for thepreparation of gold nanoparticles have previously been reported (forexample, J. Turkevich, et al., Discuss. Faraday. Soc. 1951, 11, 55-75;J. Kimling, et al, J. Phys. Chem. B 2006, 110, 15700-15707; G. Frens,Colloid & Polymer Science 1972, 250, 736-741; G. Frens, Nature (London),Phys. Sci. 1973, 241, 20-22; M. Brust et al., J. Chem. Soc., Chem.Commun. 1994, 7, 801-802; Manna, et al. (2003) Chem. Mater. 15(1):20-28; S. D. Perrault; W. C. W. Chan (2009) J. Am. Chem. Soc. 131(47): 17042; M. N. Martin et al., Langmuir 26 (10): 7410). Colloidalgold is often prepared by reduction of gold halides monodispersedparticles with a diameter of 10±60 nm were described by Frens usingsodium citrate for the reduction of HAuCl₄.

Gold hydrosol is a typical lyophobic colloid, the particles of whichbears a large negative surface charge (the surface potential is ˜50 mV)and, hence, it is stable only in very low-ionic-strength solutions. Inlyophobic systems, the dispersion medium and the dispersed phase aresubstantially different in the chemical composition and the interfacestructure, as a result of which the surface forces at the interface areuncompensated. Therefore, these systems are thermodynamically unstableand require special stabilization.

Stabilizing gold nanoparticles: a strong negative charge of the goldparticle surface provides their strong adsorption interactions withhigh-molecular-mass compounds. Sulfur and gold atoms are known to formdative bonds. Alkane thiol linkers HS(CH2)nR(R═COOH, OH or SO3H;n=11±22) are being used to achieve stronger attachment of bio-moleculesto gold particles. Interactions of these linkers with gold affordthiolates which form a monolayer on the particle surface.

In recent years, synthetic polymers, such as polyethylene glycol (PEG),polyethyleneimine, polyvinylpyrrolidone, poly-(vinyl acetate),polyamidoamine (dendrimer), polydithiafulvene, chitosan, and the like,have found application in the synthesis of mono-dispersed colloidal gold(CG). Particles formed in the presence of these polymers arecharacterized by a higher size and shape uniformity. Unfortunately, thesynthesis methods and stabilizers which have been used for producingstable gold nanoparticles did not provide the optimal gold nanoparticlesfor medical use, which needs highly stable and very concentrated metalnanoparticles in aqueous dispersion. Uses for metal nanoparticles inmedical applications have been proposed, such as, for example, in U.S.Pat. No. 6,955,639, which is directed to methods of enhancing radiationeffects with metal nanoparticles.

There still remains a need in the art for the preparation of metalnanoparticles that exhibit enhanced properties such as improvedstability and solubility, reduced toxicity, enhanced bioavailability,improved pharmacokinetics, for their use in treatment and diagnosis ofvarious health related conditions.

SUMMARY OF THE INVENTION

The present invention provides for novel surface-modified heavy metalnanoparticles, compositions comprising the same and uses thereof. Thenovel surface-modified heavy metal nanoparticles of the presentinvention exhibit improved properties as compared to other metalnanoparticle preparations. In particular, the present invention providesfor novel polyethylene glycol (PEG)-N-acetyl cysteine (NAC) surfacemodified gold nanoparticles, which exhibit improved and enhancedproperties. Further provided are methods for the preparation of thestable aqueous dispersion of surface-modified heavy metal nanoparticleswhich may be used for cancer treatment or cancer diagnosis.

In some embodiments, the present invention is based on the surprisingand unexpected finding that metal nanoparticles modified by thiolcontaining groups such as Alkane thiol linkers HS(CH2)_(n)R were foundto be unstable in protein containing solutions, and other physiologicalsolutions, as such that the nanoparticles form aggregates and lose theirnanoparticulate form and structure. Unexpectedly, the inventors foundthat the novel composition of PEG-NAC, used for surface modification ofmetal nanoparticles enables the dispersion and solubility of suchnanoparticles in protein containing solutions as well as otherphysiological solutions, such as, for example, blood and plasma. The useof NAC-PEG for surface modification provides superior results ascompared to surface modification by thiol containing groups (such asNAC) alone, which do not allow the dispersion or solubility ofnanoparticles in protein containing solutions and other physiologicalmediums. According to further embodiments, the novel PEG-NACsurface-modified heavy metal nanoparticles of the present inventionexhibit improved/enhanced properties, such as, for example: improvedsolubility in water (i.e. being highly hydrophilic); improved stability(stable for a long period of over 6 months and stable at physiologicalconditions); reduced toxicity; improved bioavailability (lower does ofthe surface-modified heavy metal nanoparticles are needed to obtain abiological effect); improved pharmacokinetic properties (such asexemplified by higher half life in blood); being biodegradable; easy andcost effective to produce (even at larger industrial scale); and thelike. In further embodiment, the improved properties of the novelsurface-modified heavy metal nanoparticles allows the preparation ofhighly concentrated preparations of the coated heavy metal nanoparticlesand further allows their production in the form of a powder. In furtherembodiments, the novel surface-modified heavy metal nanoparticles of thepresent invention allows the attachment of various drugs thereto, whichmay further be used as carriers to deliver drugs or other substances toa desired location. Thus, the novel surface-modified heavy metalnanoparticles of the present invention enable the use of higher amountsof the heavy metal, with a reduced toxicity and enhanced biologicaleffect as compared to other metal containing nanoparticles.

Thus, in some embodiments, the present invention provides asurface-modified heavy metal nanoparticle, comprising a heavy metal coreand a coating layer, the coating layer comprising at least one ligand,wherein the at least one ligand is bound to the surface of the heavymetal nanoparticle core and wherein the at least one ligand isconjugated to a polymer.

According to some embodiments there is provided a surface-modified heavymetal nanoparticle comprising: a heavy metal core and a coating layer,the coating layer comprising at least one ligand conjugated topolyethylene glycol (PEG), wherein the at least one ligand is selectedfrom N-acetyl cysteine (NAC), albumin, cysteine, methionine,glutathione, amino thiols, thio-carboxylic acids, ammonia, amines,diamines or any combination thereof; and wherein the at least one ligandis bound to the surface of the heavy metal nanoparticle core. In someembodiments, the ligand is N-acetyl cysteine (NAC). In some embodiments,the at least one ligand is covalently bound to the surface of the heavymetal nanoparticle core.

In some embodiments, the heavy metal is selected from gold, goldspecies, silver, platinum, iron, copper, nickel, palladium, iridium,titanium or lead. In some embodimentns, the heavy metal is gold species.In further embodiments, the gold species may be selected from AuCl₃,AuF₃, AuBr₃, HAuCl₄ or MAuCl₄, wherein M represents an alkali metalcation.

In further embodiments, the nanoparticle is of the size from about 0.5nm to about 400 nm.

In some embodiments, the surface-modified heavy metal nanoparticles arein a substantially dry powder form.

In some embodiments, there is further provided an aqueous dispersion ofthe surface-modified heavy metal nanoparticles. The particles may bedispersed in water or in a buffer. In some embodiments, the buffer maybe at a pH of between about 4.5 to about 8.

In some embodiments, there is provided a surface-modified goldnanoparticle comprising: a gold species metal core and a coating layer,the coating layer comprising N-acetyl cysteine (NAC) ligand conjugatedto polyethylene glycol (PEG), wherein the N-acetyl cysteine (NAC) ligandis bound to the surface of the gold species nanoparticle core.

According to some embodiments, there is provided a compositioncomprising N-acetyl cysteine (NAC) conjugated to polyethylene glycol(PEG), for the surface modification of metal nanoparticles.

According to some embodiments, there is provided a process for thepreparation of surface-modified heavy metal nanoparticles comprising thesteps of:

adding at least one ligand conjugated to poly ethylene glycol (PEG) to amixture comprising metal nanoparticles, wherein the at least one ligandbinds to the surface of the heavy metal nanoparticles core, yieldingsurface-modified heavy metal nanoparticle, wherein the ligand ligand isselected from N-acetyl cysteine (NAC), albumin, cysteine, methionine,glutathione, amino thiols, thio-carboxylic acids, ammonia, amines,diamines or any combination thereof. In some embodiments, the ligand isN-acetyl cysteine (NAC). In some embodiments, the mixture is preparedby:mixing at least one surfactant with at least one organic solvent in awater solution to yield an emulsion; andadding to the emulsion of step (a) a solution of heavy metal species andat least one reducing agent, to yield reduced metal nanoparticles.

In some embodiments, the heavy metal is a gold species, selected fromAuCl3, AuF3, AuBr3, HAuCl4 or MAuCl4, wherein M represents an alkalimetal cation. In some embodiments, the heavy metal nanoparticles are ofa size of from about 0.5 to about 400 nm.

In some embodiments, the process further comprises drying the aqueousphase, yielding surface-modified heavy metal nanoparticles in a dryform.

In some embodiments, the process comprises dispersing the heavy metalnanoparticles in water or in a buffer. The buffer may be at a pH betweenabout 4.5 to about 8.

In some embodiments, the at least one surfactant comprises at least onefatty acid. In some embodiments, the at least one fatty acid is oleicacid.

In some embodiments the organic solvent is ethanol. In some embodimentsthe at least one reducing agent is any one of ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium citrate, sodium borohydride orlithium borohydride. In some embodiments, the reducing agent is ascorbicacid.

In some embodiments, the process further comprises adding at least onesecond organic solvent. In some embodiments, the second organic solventis selected from hexane, cyclohexane, chloroform, diethyl ether, ethylacetate and toluene. In some embodiments,

In some embodiments, there is provided a pharmaceutical compositioncomprising the surface-modified heavy metal nanoparticles. Thepharmaceutical composition may further comprise at least one ofpharmaceutically acceptable additives, carriers, buffers, stabilizers orexcipients. In some embodiments, the pharmaceutical composition issuitable for oral administration, injection or infusion. In someembodiments, the pharmaceutical composition is for use in medicaltreatment or medical diagnosis. In some embodiments, the pharmaceuticalcomposition is for use in the treatment of diagnosis of malignantdisorders, wherein the malignant disorder is any one of carcinoma,sarcoma, germ cell tumors or blastoma. In further embodiments, thetreatment or diagnosis is in vitro or in vivo.

In additional embodiments, there is provided an injectable solutioncomprising the pharmaceutical composition, which comprises thesurface-modified heavy metal nanoparticles. In further embodiments,there is provided a sterile syringe comprising the injectable solution.

In some embodiments, there is provided a kit comprising an aqueousdispersion of the surface-modified heavy metal nanoparticles. In someembodiments, the dispersion comprises water or a buffer at pH values ofbetween about 4.5 to about 8, or pharmaceutical composition comprisingthe same; means for administering the aqueous dispersion orpharmaceutical composition into a patient; and instructions for use.

In some embodiments, there is provided a kit comprising thesurface-modified heavy metal nanoparticles in a dry form; an aqueoussolution for dispersing the surface-modified heavy metal nanoparticles.In some embodiments, the dispersion comprises water or a buffer at pHvalues of between 4.5 and 8; means for administering the aqueousdispersion into a patient; and instructions for use.

In further embodiments, there is provided a method of treatment ordiagnosis of a malignant disorder comprising the steps of administratinga subject in need a therapeutically effective amount of thesurface-modified heavy metal nanoparticles or of a pharmaceuticallycomposition comprising the same. The administration may be performed byoral, infusion or injection administration routes.

In additional embodiments, there is provided an N-acetyl cysteine(NAC)-polyethylene glycol (PEG) conjugate for use in surfacemodification of metal nanoparticles, wherein the N-acetyl cysteine (NAC)is capable of bounding to the surface of the metal nanoparticle core. Insome embodiments, the metal is gold species.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIGS. 1A-1B are Transmission Electron Microscopy (TEM) images showing afield of an aqueous dispersion of surface coated gold nanoparticles asindicated by the appearance of black dots at a magnification of 100 nm(FIG. 1A) and 50 nm (FIG. 1B). The images indicate that thenanoparticles are separate and have a size smaller than or equal to 10nm.

FIG. 2. shows ¹H-NMR spectrum of PEG-NAC molecule (conjugate/compound)which is used as surface coating for heavy metal nanoparticles.

FIGS. 3A-D show graphs of change in mean body weight (g) over time (daysafter injection) of mice groups following multiple intravenous (IV)injection of varying concentration of gold nanoparticles. FIG. 3A—changein body weight of group I mice (treated with 100 mg/kg goldnanoparticles every other day, for six days). FIG. 3B-change in bodyweight of group II mice (treated with 100 mg/kg gold nanoparticles, iv,for five days, two days off and 5 more days (5/2/5)). FIG. 3C-change inbody weight of group III mice (treated with 150 mg/kg goldnanoparticles, iv, 5/2/5). FIG. 3D-change in body weight of group IVmice (treated with 200 mg/kg gold nanoparticles, iv, 5/2/5). Nomortality occurred in the animals treated with the GNPs prior to thescheduled termination, carried out 26 days post-dosing;

FIG. 3E. A graph of percent body weight changes over time (days) inhealthy female nude mice following multi dose i.v. of 200 mg/mlPEG-NAC-gold nanoparticles alone (G1) or in combination with irradiation(G2).

FIG. 4. Pictograms of cell culture medium dish containing PEG-NACsurface coated gold nanoparticles (left hand dish) or cell culturemedium dish containing NAC surface coated gold nanoparticles (right handdish). The cell culture medium dish containing PEG-NAC surface coatedgold nanoparticles appears clear as compared to cell culture mediumcontains the same concentration of NAC surface coated gold nanoparticle,in which, formation of GNPs-protein conjugates is observed.

FIG. 5A. Pictogram of NAC-gold nanoparticles in Human blood, formationof protein-GNPs conjugates is shown.

FIG. 5B. Pictogram of PEG-NAC-gold nanoparticles in Human blood. Noformation of protein-GNP conjugates is shown.

FIG. 6A. A graph showing the pharmacokinetics of NAC surface-modifiedgold nanoparticles in plasma or in tumors of mice bearing tumors. Thegraph shows the concentration of gold in plasma or in tumor of mice thatwere injected with the NAC surface-modified gold nanoparticles (hoursafter injection).

FIG. 6B. A graph showing the pharmacokinetics of PEG-NACsurface-modified gold nanoparticles in plasma or in tumors of micebearing tumors. The graph shows the concentration of gold in plasma orin tumor of mice bearing tumors that were injected with the PEG-NACsurface-modified gold nanoparticles (hours after injection);

FIG. 6C. A graph showing the percent injected dose (%) per gram tumorover time (days after injection) in tumor, of PEG-NAC surface modifiedgold nanoparticles (tumor AuPEG-GNPs) or of NAC-surface modified goldnanoparticles (“regular GNPs”).

FIG. 6D. A graph showing the percent injected dose (%) per gram overtime (days after injection) in plasma, of PEG-NAC surface modified goldnanoparticles (AuPEG-GNPs) or of NAC-surface modified gold nanoparticles(“regular GNPs”).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based on results of a research thatsurprisingly provides stable and non-toxic aqueous dispersions ofsurface modified heavy metal nanoparticles, present at highconcentration in the aqueous dispersions, which may be systemicallyadministered to a subject and may exhibit improved properties, such as,reduced toxicity, improved stability, enhanced bioavailability, improvedpharmacokinetics, and the like.

The inventors have developed a novel aqueous dispersion ofsurface-modified metal nanoparticles and a methodology allowing theirpreparation. In particular, the inventors have developed novelsurface-modified gold nanoparticles, wherein the surface modificationcomprises N-acetyl cysteine (NAC) ligand conjugated to polyethyleneglycol (PEG), wherein the N-acetyl cysteine (NAC) ligand is bound to thesurface of the gold species nanoparticle core. Unexpectedly, suchmodification provides improved and enhanced properties to thenanoparticles. As exemplified herein, the improved properties includesuch properties as, but not limited to: improved solubility in water(i.e. being highly hydrophilic); improved stability (stable for a longperiod of over 6 months and stable at physiological conditions); reducedtoxicity; improved bioavailability (lower does of the surface-modifiedheavy metal nanoparticles are needed to obtain a biological effect);improved pharmacokinetic properties (such as exemplified by higher halflife in blood); being biodegradable; easy and cost effective to produce(even at larger industrial scale); and the like.

The inventors have found that the formation of a stable aqueousdispersion of surface-modified gold nanoparticles is possible bypreparing an intermediate-state compound, namely reduced goldnanoparticles in an emulsion environment, prior to the addition of acoating ligand thereby obtaining surface-modified gold nanoparticles.The preparation of the aqueous dispersions of gold nanoparticles via theabove mentioned intermediate step assists in obtaining stable goldnanoparticles which can be stably dispersed in aqueous solutions,particularly at significant concentrations.

The surface-modified metal nanoparticles disclosed herein are present inthe aqueous dispersions of the invention at high concentrations and arestable at physiological pH of 7.3-7.4 or in a living cell, tissue,organ, and in in vivo or in in vitro environments. In addition, thenanoparticles may be used by systemic administration as therapeuticmeans or for diagnostic purposes.

For example, the aqueous dispersions of the surface-modified metalnanoparticles contained at high concentrations, may significantlyimprove the radiation therapy of malignant disorders possibly by theability of the gold nanoparticles to absorb significantly more of thetreatment radiation than other light atoms surrounding them and theirability to emit secondary radiation in the form of electrons and lowenergy photons, this secondary radiation being highly effective indestroying cancer cells.

The aqueous dispersions of surface-modified gold nanoparticles areproduced by novel preparation methods as described below.

The present invention encompasses several aspects as will be describedbelow, and may be summarized as follows: surface-modified metalnanoparticles, an aqueous dispersion of surface-modified metalnanoparticles, a method for their preparation, pharmaceuticalcompositions comprising the same and their uses in medical treatment ordiagnosis.

Thus, in accordance with one aspect, there is provided surface-modifiedheavy metal nanoparticle, comprising a heavy metal core and a coatinglayer, the coating layer comprising at least one ligand, wherein the atleast one ligand is bound to the surface of the heavy metal nanoparticlecore through covalent bonds.

The term “nanoparticle” is used herein to denote any microscopicparticle smaller than about 400 nm in diameter, wherein eachnanoparticle behaves as a complete unit in terms of the transportcharacteristics and physical and chemical properties.

As used herein the term “about” refers to ±10% of the indicated value.

The term “heavy metal” is used herein to denote a member of a subset ofelements that exhibit metallic properties, which may include transitionmetals, metalloids, lanthanides, or actinides.

In some embodiments, the heavy metal may be for example any one of gold,platinum, silver, iron, copper, nickel, palladium, iridium, titanium orany other heavy metal. According to one embodiment, the metal is iron.

According to some embodiments, the metal is gold. According to furtherembodiments, the gold is a gold species selected from AuCl3, AuF3,AuBr3, HAuCl4 or MAuCl4, wherein M represents an alkali metal cation.

As used herein the term “surface-modified heavy metal nanoparticles”refers to metal nanoparticles, comprising a coating layer on theirsurface, whereby the coating layer modifies the nanoparticles surfaceand wherein the coating layer comprises at least one ligand. In someembodiments, the ligand may be further conjugated to a polymer.

As used herein the term “PEG-NAC surface modified heavy metalnanoparticles” is directed to heavy metal core nanoparticle and acoating layer, the coating layer comprising N-acetyl cysteine (NAC)ligand conjugated to polyethylene glycol (PEG), wherein the N-acetylcysteine (NAC) ligand is bound (covalently or non covalently) to thesurface of the heavy metal nanoparticle core. In exemplary embodiments,the heavy metal is a gold species.

As used herein, the terms “PEG-NAC compound(s)”, “PEG-NAC conjugate(s)”or “PEG-NAC molecule(s)” may interchangeably be used and are directed toa compound comprising polyethylene glycol (PEG) conjugated to N-acetylcysteine (NAC).

As used herein the term “NAC surface modified heavy metal nanoparticles”is directed to heavy metal core nanoparticle and a coating layer,comprising N-acetyl cysteine (NAC) ligand, which is not conjugated topolyethylene glycol (PEG). The N-acetyl cysteine (NAC) ligand is bound(covalently or non covalently) to the surface of the heavy metalnanoparticle core. In exemplary embodiments, the heavy metal is a goldspecies.

As used herein, the term “GNP(s)” is directed to gold nanoparticles.

As used herein the coating of the nanoparticles surface with the atleast one ligand is for example coating at least 60%, at least 70%, atleast 80%, at least 90% of the outer surface of the core of thenanoparticle.

The term “ligand” as used herein is any organic ligand which is solublein water and is capable of binding (covalently or non-covalently) thesurface of the metal nanoparticles core and thus modifying the surfaceof the nanoparticles by coating the surface, thereby formingsurface-modified metal nanoparticles.

In some embodiments, the ligand is at least one of N-acetyl cysteine,albumin, or amino acids such as for example, but not limited tocysteine, methionine and glutathione, amino thiols, thio-carboxylicacids, ammonia and amines, diamines or any combination thereof, or anyligand capable of binding gold nanoparticles.

In one embodiment, the ligand may be N-acetyl cysteine. In oneembodiment, the ligand may be cysteine.

In accordance with the present disclosure, the ligand may have at leastone free thiol group which may bind for example to the metalnanoparticles via the interaction of the any one of the thiol groups ofthe ligand and the metal atoms.

According to some embodiments, the ligand may be conjugated to apolymer. In one embodiment, the polymer is polyethylene glycol (PEG).

According to one embodiment, the surface-modified metal nanoparticlesare of a size of from about 0.5 nm to about 400 nm.

In some embodiments, the size is smaller than 100 nm, for examplesmaller than 90 nm, smaller than 80 nm, smaller than 70 nm, smaller than60 nm, smaller than 50 nm, smaller than 40 nm, smaller than 30 nm,smaller than 20 nm, or smaller than 10 nm. In some embodiments, thenanoparticles are of a size smaller than 10 nm.

In some embodiments, the surface-modified heavy metal nanoparticle maybe in a substantially dry powder form. The dry powder may be storedunder appropriate conditions for example under vacuum conditions attemperatures of between about 2° C. to about 8° C. for long periods oftime, possibly until further use.

In some embodiments, the present invention provides an aqueousdispersion of surface-modified heavy metal nanoparticles, dispersed inwater or in a buffer at pH between about 4.5 to about 8, for example, ina physiological buffer at pH between about 7.3 to about 7.4.

According to some embodiments, an aqueous dispersion of surface-modifiedheavy metal nanoparticles may be obtained by dissolving the dry powdercomprising the surface-modified heavy metal nanoparticle, in water or ina buffer solution at a pH between about 4.5 to about 8, for example, atphysiological pH of about 7.3 to about 7.4.

The surface-modified nanoparticles dispersions of the invention arechemically as well as physically storage-stable for at least 3 month,and even for a period of 6 months, when stored at appropriate storageconditions of 2° C.-8° C.

The “stability” in the context of the present disclosure may bedetermined by various chemical and/or physical methods, and is to betaken to mean that no significant formation of aggregates orprecipitation are observed. Under these storage conditions, no formationof aggregates or precipitation is observed. In some embodiments,“stability” is directed to the preservation of the nano size of theparticles.

In general, it can be appreciated that the stabilization depends onsolubility of the ligand in a dispersion medium, the ability ofnanoparticles to bind the ligand on their surface and the degree of thesurface coverage by the ligand.

The aqueous dispersions of surface-modified metal nanoparticles of thepresent invention may be used in cancer treatment. As shown in theexemplary embodiments, when cancer cells were incubated in mediumcomprising the aqueous dispersions of gold nanoparticles, the goldnanoparticles were delivered into cancer cells in an amount which isdependent on the nanoparticles concentration in the incubation mediumand on the incubation time, with no observed toxicity side effects.

The present disclosure provides the method for the preparation ofsurface-modified heavy metal nanoparticle comprising the steps of:

-   (a) mixing at least one surfactant with at least one organic solvent    in a water solution to yield an emulsion;-   (b) adding to the emulsion of step (a) a solution of heavy metal    species and at least one reducing agent, to yield reduced metal    nanoparticles;-   (c) adding at least one ligand to the mixture of step (b), wherein    the at least one ligand binds the surface of the heavy metal    nanoparticles core, yielding surface-modified heavy metal    nanoparticle.

The method may further include an additional step (d) which comprisesseparating the inorganic phase which contains the heavy metalnanoparticles from the organic phase.

In some embodiments, the at least one organic solvent of step (a) is awater miscible organic solvent. In some embodiments, step (a) the atleast one surfactant is mixed with at least one first water-miscibleorganic solvent in a water solution to yield an emulsion.

The term “surface-modified heavy metal nanoparticle” is directed toheavy metal nanoparticles coated with a ligand, wherein the ligand isbound to the surface of the metal nanoparticles, thereby modifying saidsurface, and wherein the “heavy metal” may be any one of gold, platinum,silver, iron, copper, nickel, palladium, iridium, titanium or any otherheavy metal. In one embodiment, the metal is gold. In some embodiments,the ligand may be further conjugated to another substance, such as apolymer.

In accordance with the first step of the process (a), an emulsion isobtained upon mixing at least one surfactant with at least one firstwater-miscible organic solvent in an alkaline base or water solution.

The term “emulsion” is to be understood in the context of the presentinvention as a mixture of two or more immiscible (unblendable) liquids.Emulsions are made up of a dispersed and a continuous phase; theboundary between these phases is called the interface. Microemulsionstend to appear clear due to the small size of the disperse phase.

In the context of this invention, the term “surfactant” may be referredto a compound that lowers the surface tension of a liquid.

In some embodiments, the surfactant may comprise a fatty acid, whereinthe fatty acid may be for example a long-chain saturated fatty acid or amono- or poly-unsaturated fatty acid.

In some embodiments, the fatty acid may be selected for example fromoleic acid, linoleic acid or erucic acid, palmitoleic acid, sapienicacid myristoleic acid. According to some embodiments the fatty acid isoleic acid

According to some embodiments, the at least one surfactant may comprisea mixture of fatty acids. According to some other embodiments, themixture of fatty acids may predominantly comprise oleic acid. In somespecific embodiments the mixture of fatty acids comprises 65% wt. ofoleic acid.

In the context of this invention, the at least one “first water-miscibleorganic solvent” is to be referred to any organic solvent which uponmixing with water forms a substantially uniform mixture. Thewater-miscible organic solvent according to the invention may be ethanolor acetone. According to some embodiments, the water-miscible organicsolvent may be ethanol.

The term “an alkaline solution” in the context of the present inventionis to be referred to a as a solution of a soluble base, wherein “base”is defined by the general chemistry definition, wherein a base is asubstance that can accept hydronium ions.

The base used according to the present invention may be any alkalinebase for example sodium hydroxide solution or potassium hydroxidesolution or any other alkaline metal hydroxide but other bases may bealso suitable including for example ammonia. In some embodiments, thebase is sodium hydroxide.

The solution comprising the at least one surfactant, wherein thesurfactant may be for example fatty acid or a mixture of fatty acids, atleast one first water-miscible organic solvent may be mixed in analkaline solution to form an emulsion under conditions which partiallydissolve the fatty acids.

Mixing may be for example using a magnetic stirrer for a suitable amountof time for example for about 5 minutes at a temperature such as roomtemperature.

In accordance with the second step of the process (b), a solution ofheavy metal species and at least one reducing agent are added to theemulsion detailed above to yield reduced metal nanoparticles.

In some embodiments, the “heavy metal” may be any metal species asdefined and detailed above. In some embodiments, the metal is gold.

The “ionic metal species” as used herein is considered to be anysubstance or chemical entity that contains or can generate (andtherefore a precursor) metal ions

According to some embodiments wherein the metal is gold, “ionic goldspecies” may be selected from AuCl3, AuF3, AuBr3, HAuCl4 or MAuCl4,wherein M represents an alkali metal cation, for example sodium orpotassium cation. According to some other embodiments, the ionic goldspecies is HAuCl4. HAuCl4 is thus a precursor ionic species, whichyields [AuCl3]-ions.

According to some other embodiments “ionic metal species” may beselected from FeCl2, FeSO4, FeCl3.

The at least one “reducing agent” used herein is an agent capable ofreducing the ionic metal species within the emulsion. In someembodiments, the reducing agent is organic or inorganic. Non-limitingexamples of such reducing agents may be for example any one of ascorbicacid, ethylene diamine tetraacetic acid (EDTA), sodium citrate in thepresence or absence of tannin, sodium borohydride, borohydride in amixture with sodium citrate or EDTA and cyanoborohydride, hydrazine,sodium diphenylaminosulfonate or lithium borohydride and anycombinations thereof.

According to some other embodiments, the reducing agent is ascorbicacid.

In an embodiment where the metal is gold, the reducing agent may beadded for example at a 2:1 molar ratio relative to the ionic goldspecies.

Alternative reducing methods which may be used in accordance with thepresent invention may be for example irradiation methods such asultrasonic, UV irradiation or pulse or laser radiolysis.

In an embodiment where the metal is gold, the ionic metal species may beadded during mixing, and may optionally result in a yellowish solution.Upon addition of a solution comprising the at least one reducing agent,the solution color may change to red-wine purple color, possiblyindicating completion of the reduction of the ionic gold species.

In the context of the present application, it is to be understood thataddition of metal species within the emulsion may provide means tocontrol the size of the formed nanoparticles and hence enables theformation of nanoparticles of a predetermined size.

In accordance with the third step of the process (c), at least oneligand is added to the mixture above.

The term “ligand” as used herein is as detailed above and may be anyorganic ligand which is soluble in water and is capable of coating thesurface of the metal nanoparticles by binding (covalently ornon-covalently) to the metal thereby forming surface-modified metalnanoparticles.

The covalent binding of the at least one ligand having at least one freethiol group may be for example via the interaction of the any one of thethiol groups of the ligand and the metal atoms.

In some embodiments, the ligand is at least one of N-acetyl cysteine,albumin and amino acids, for example cysteine and methionine,glutathione, amino thiols, thio-carboxylic acids, ammonia and amines,diamines or any combinations thereof or any ligand capable of bindingmetal nanoparticles.

In some embodiments, the ligand is N-acetyl cysteine. In someembodiments, the ligand is cysteine. The molar ratio between the metaland the ligand may be 1:4, preferably 1:6, more preferably 1:8.

In accordance with the invention, the ligand may be conjugated to apolymer. The polymer according to the present disclosure is an inert anda nontoxic polymer. Polymers typically used as include, without beinglimited thereto: polyethylene glycol (PEG), polyethyleneimine,polyvinylpyrrolidone, poly-(vinyl acetate), polyamidoamine (dendrimer),polydithiafulvene, chitosan.

In some embodiments, the polymer may be a linear polymer, for examplePEG.

According to the present disclosure, conjugation of PEG to the at leastone ligand may be performed before the addition of the at least oneligand to the mixture as detailed above.

According to some embodiments, the surface modification of the metalnanoparticles is a compound of N-acetyl cysteine (NAC) conjugated topolyethylene glycol (PEG). In some embodiments, the molar ratio betweenthe metal nanoparticles and NAC-PEG compound may be at the range ofabout 1:1 to 1:20. For example, in some embodiments, the molar ratiobetween the metal nanoparticles and NAC-PEG compound may be 1:1. Forexample, in some embodiments, the molar ratio between the metalnanoparticles and NAC-PEG compound may be 1:2. For example, in someembodiments, the molar ratio between the metal nanoparticles and NAC-PEGcompound may be 1:4. For example, in some embodiments, the molar ratiobetween the metal nanoparticles and NAC-PEG compound may be 1:8. Forexample, in some embodiments, the molar ratio between the metalnanoparticles and NAC-PEG compound may be 1:10. For example, in someembodiments, the molar ratio between the metal nanoparticles and NAC-PEGcompound may be 1:20.

According to some embodiments, there is provided a NAC-PEG compound. Insome embodiments, the NAC-PEG compounds may be used for the surfacemodification of metal nanoparticles.

In accordance with the present invention, the aqueous dispersion ofsurface-modified heavy metal nanoparticles may be conjugated to a secondmolecule for example to an antibody, wherein the antibody may assist intargeting the metal nanoparticles for example to a site of interest asfor example in diagnosis. In addition, the nanoparticles of the presentinvention may be conjugated to a visually or otherwise detectablemoiety, for example a chromophore or fluorophore.

Following the addition of the at least one ligand, the solution may bemixed for a suitable periods of time for example but not limited toabout 30 minutes, more preferably for more than an hour.

The addition of the at least one ligand to the mixture comprisingreduced metal nanoparticles may result in the formation of water-solublesurface-modified metal nanoparticles.

According to some embodiments, the pH may be adjusted during theaddition of the at least one ligand to a basic pH, preferably to a pHvalue above pH of 9, 9.5, 10, preferably above pH 9. This may allow mostof the fatty acid to be converted to fatty acid salt, which in turnallows the ligand to bind the gold nanoparticles.

The pH may be adjusted according to the invention using for example bytitration with any alkaline base, possibly sodium hydroxide or potassiumhydroxide.

According to some further embodiments, at least one second organicsolvent may be added to the solution comprising water-solublesurface-modified gold nanoparticles and the pH may be thereafteradjusted to an acidic pH. The solution may be stirred for example for atleast an hour.

According to the present invention, the “second organic solvent” is anyorganic solvent which is immiscible with water which at an acidic pHdissolves the fatty acid. The addition of this solvent tends to causeseparation of the two phases into un-emulsified distinct phases, therebyseparating the organic phase from the aqueous phase.

In some embodiments, the second organic solvent is hexane, cyclohexane,diethyl ether, pentane, cyclopentane, chloroform, ethyl acetate ortoluene. According to some embodiment, the second organic solvent ishexane.

According to some embodiments, the second organic solvent is added andthe pH is adjusted to an acidic pH, for example, lower than pH 7, lowerthan pH 6.5, lower than pH 6, lower than pH 5.5, lower than pH 5, lowerthan pH 4.5, lower than pH 4, lower than pH 3. In some embodiments, thepH is lower than pH 6.5. The pH may be adjusted using any acid, forexample HCl.

The addition of a second organic solvent and adjusting the pH to anacidic pH enables the phase separation, wherein the emulsion isseparated into an aqueous phase and an organic phase.

The aqueous phase may comprise the surface-coated metal nanoparticles,wherein the organic phase may comprise the organic solvents and fattyacids.

According to some embodiments, the at least one ligand may be also addedto the reaction mixture following the addition of the second organicsolvent. According to some embodiments, the at least one ligand willbind free sites on the surface of the metal nanoparticles. According tothis embodiment, the ligand may be, for example, albumin that may beadded and the mixture may be allowed to stir, for example, for 30 min ona magnetic stirrer.

Following the phase separation, the organic phase may be separated fromthe aqueous phase for example by suitable means, such as, for example byusing a separation funnel.

In accordance with the present invention, the aqueous phase may befurther dried. Drying of the aqueous phase according to the presentinvention may be, for example, by evaporation, vacuum drying,freeze-drying (lyophilization), desiccation or any other suitabletechnique of removing water residues or moisture and obtaining a productwhich is solid, for example, in a powder form.

In some embodiments, drying may comprise evaporation of traces of thefirst water-miscible organic solvent, resulting in a dried powder ofsurface-modified metal nanoparticles.

In some embodiments, the dry powder may be dissolved in water or in abuffer solution at a pH between about 4.5 to about 8, preferably atphysiological pH of 7.3-7.4. Dissolving the dry powder yields an aqueousdispersion of surface-modified heavy metal nanoparticles.

In some embodiments, the aqueous dispersion of metal nanoparticlescomprises metal nanoparticles at the size of from about 1 nm to about100 nm, from about 1 nm to 10 nm, from about 10 nm to 20 nm, from about20 nm to 30 nm, from about 30 nm to 40 nm, from about 40 nm to 50 nm,from about 50 nm to 60 nm, from about 60 nm to 70 nm, from about 70 nmto 80 nm, from about 80 nm to 90 nm, from about 90 nm to 100 nm. In someembodiments, the metal nanoparticles are of a size smaller than 10 nm.

The metal nanoparticles of the present invention are dispersed in anaqueous solution at concentrations of 0.001 μM-1M, for example atconcentrations greater than 0.005M, greater than 0.01 M, greater than0.05M, greater than 0.1 or sometimes greater than 0.5M.

Preparation of metal nanoparticles may be performed by using thetwo-phase micro emulsion method (for example as described by M. Burst etal., J. Chem. Soc., Chem. Commun. S, 1994, 7, 801-802). Accordingly,metal-containing reagents are transferred from an aqueous to an organicphase. After the addition of a surfactant solution, a micro emulsion isformed. The reduction reaction proceeds in a dispersed phase in whichthe drop size is at most 100 nm. As a result, virtually mono dispersednanoparticles are formed. In micro emulsion methods of synthesis ofnanoparticles, alkane thiols are often added to the reaction solution,for the stabilization of the particles. The final gold nanoparticles aredispersed in organic phase, stabilized with alkane thiol.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising surface-modified heavy metal nanoparticles asdisclosed herein. In some embodiments, the pharmaceutical compositionand formulations may include sterile aqueous solutions which may alsocomprise at least one of pharmaceutically acceptable additives such as,but not limited to, penetration enhancers, carrier compounds, buffers,stabilizers, diluents or other pharmaceutically acceptable carriers orexcipients.

According to some embodiments, the pharmaceutical composition may beused in medical treatment or in medical diagnosis.

The pharmaceutical compositions of the present disclosure may be usedfor example in the medical diagnosis of malignant disorders. “Medicaldiagnosis” as used herein refers to the determination of the identity ofa possible disease or disorder. Diagnosis may be based on combinationsof signs, symptoms and test results. In accordance with the presentdisclosure, the pharmaceutical composition may be used for example incancer detection for example as probes for molecular imaging,photoactive agents for optical imaging, contrast enhanced agent incomputer tomography (CT) or in magnetic resonance imaging (MRI) or anymethod capable detecting cancer and providing evaluation thereto.

The pharmaceutical compositions of the invention may also compriseadditional therapeutically active agents.

According to some embodiments, the pharmaceutical composition may besuitable for oral administration, injection or infusion.

The pharmaceutical compositions of the present disclosure may be used,for example, in the treatment of malignant disorders or cellproliferative disorders, such as, for example malignancies, that may be,for example solid or non-solid tumors. In some embodiments, thepharmaceutical compositions of the present disclosure may be used, forexample, in the treatment of non-malignant disorders associated withincreased, enhanced or abnormal proliferation of cells.

In some embodiments, the malignant disorders may be, for example, solidtumors such as carcinoma, sarcoma, melanoma, germ cell tumors orblastoma.

In some embodiments, the malignant disorders may be for example tumorsin lip and oral cavity, pharynx, larynx, paranasal sinuses, majorsalivary glands, thyroid gland, esophagus, stomach, small intestine,colon, colorectum, anal canal, liver, gallbladder, extrahepatic bileducts, ampulla of Vater, exocrine pancreas, lung, pleural mesothelioma,bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin,breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopiantube, gestational trophoblastic tumors, penis, prostate, testis, kidney,renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid,carcinoma of the conjunctiva, malignant melanoma of the conjunctiva,malignant melanoma of the uvea, retinoblastoma, carcinoma of thelacrimal gland, sarcoma of the orbit, brain, spinal cord, vascularsystem, hemangiosarcoma and Kaposi's sarcoma.

In some embodiments, the malignant disorders may comprise, for example,non-solid tumors such as leukemia or lymphomas.

In some embodiments, the medical treatment may be, for example,radiation therapy of malignant disorders or any treatment in combinationwith radiation therapy. Accordingly, pharmaceutical compositioncomprising the aqueous dispersion of metal nanoparticles may be used inradiation therapy to enhance the selectivity in treatment of malignantdisorders.

Radiation therapy is to be understood as a therapy that uses high energyradiation sources such as but not limited to X-rays, gamma rays, andcharged particles for cancer treatment by affecting the cancer cellsviability.

The radiation may be delivered by an external beam radiation sourceapparatus, synchrotron radiation sources, or alternatively by aninternal radiation source such as a radioactive material deliveredwithin the body to a location which is in close proximity to the cancercells, such as brachytherapy.

In other embodiments, the pharmaceutical composition of the inventionmay be used in combination with any hyperthermia treatment for examplein combination with laser, UV, RF, ultrasound and any other source ofhyperthermia used.

It is to be understood that the aqueous dispersion of surface-modifiedmetal nanoparticles may be themselves used in therapeutically formedical treatment or medical diagnosis or alternatively in combinationwith radiation or in combination with any treatment methodconventionally used.

According to some embodiments, the pharmaceutical composition may beadministered to a patient, for example, by systemic administration ortopical administration.

The term “systemic administration” is to be referred as a route ofadministration comprising enteral administration comprising for exampleoral, rectal, sublingual or buccal administration or parentaladministration which may comprise for example piercing the skin ormucous membrane.

The term “topical administration” is to be referred as a route ofadministration comprising application to the surface of a body part.

In some embodiments, the route of administration may involve, forexample, intravenous (i.v.), intramuscular (i.m.), intraperitoneal(i.p.), transdermal, transmucosal, intra-tumor, intragastric, intranasalor orally for example by tablets, capsules, lozenges or drops, ortopical administration for example by creams, dermal patches and thelike or any combination thereof.

In some embodiments, the pharmaceutical composition may be delivered tothe circulation through the digestive system, via intragastric or oraladministration.

In some embodiments, the pharmaceutical composition may be administratedby injection or infusion.

In some embodiments, the pharmaceutical composition may be administereddirectly to the target of interest, for example, for use in radiationtherapy. In some embodiments, the administration may be in closeproximity to the tumor to be treated, for example, by injection.

It is to be understood that the aqueous dispersion of surface-modifiedmetal nanoparticles may be administered themselves as described abovefor the pharmaceutical composition.

As can be understood herein the treatment as described according to thepresent invention may be achieved by using an effective amount of theaqueous dispersion of surface-modified metal nanoparticles orcompositions comprising the same.

The terms “effective amount”, “therapeutically effective amount” or“dosing” are used herein interchangeably and mean any amount necessaryto achieve a selected result, which present may involve the amount ofmetal nanoparticles necessary for treating cancer or proliferativemalignant or non-malignant disorders, specifically, killing thecancerous or otherwise abnormal cells.

The therapeutic effective amount, or dosing, is dependent on severityand responsiveness of the disease state to be treated, with the courseof treatment lasting one hour to several hours, or until a cure iseffected or a diminution of the disease state is achieved. Dosing may beprovided to a person in need in a regimen comprising dosing of one dailyor multiple daily administrations.

Persons of ordinary skill can readily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of the aqueous dispersion of thesurface-modified gold nanoparticles of the invention, or compositionscomprising thereof, and can generally be estimated based on EC50, foundto be effective in in vitro cellular models, ex vivo organ or tissuemodel as well as in in vivo animal models. Persons of ordinary skill inthe art can easily estimate repetition rates for dosing based onmeasured residence times, concentrations which may be affected forexample by age, sex and weight of the subject in need.

In accordance with the present disclosure, when used in a combinationtherapy, dosing may be adjusted accordingly, for example radiationtherapy or any additional therapy used.

The terms “disease”, “disorder” and “condition” are used hereininterchangeably.

As used herein to describe the present invention, “malignant”, “cancer”,“tumor”, “malignancy”, “proliferative malignant”, “hyperproliferative”all relate equivalently to a hyperplasia of a tissue or an organ.

The terms “treat”, “treating” or “treatment” as used herein meansameliorating one or more clinical indicia of disease activity in vivo ina patient having cancer or a proliferative malignant or non-malignantdisease. “Treatment” refers herein to therapeutic treatment.

In some embodiments, treatment or diagnosis of malignant disorders usingthe aqueous dispersion of surface-modified metal nanoparticles orpharmaceutical composition comprising the same may be performed in vitroor in vivo.

As used herein, the term “in vivo” may refer to an in vivo treatment orin vivo diagnosis and is to be understood as a process that takes placewithin a living organism. The term in vivo when referring to diagnosisin vivo is also to be understood as any diagnosis which may be performedin an invasive or in a noninvasive manner such as but not limited toMRI, CT in a living organism.

The term “in vitro” encompasses any treatment or diagnosis which isperformed on a body fluid such as, blood, urine, saliva or cerebrospinalfluid (CSF), organ, or tissue which can be extracted from a patient inneed.

In another aspect of the present invention, there is provided a use ofthe surface-modified heavy metal nanoparticles as described above in thepreparation of a pharmaceutical composition.

In some embodiments, the pharmaceutical composition may be used formedical treatment or medical diagnosis as described above.

In yet another aspect of the present invention there is provided aninjectable solution comprising the aqueous dispersion ofsurface-modified heavy metal nanoparticles or pharmaceutical compositioncomprising the same as described above. In some embodiments, there isprovided a sterile syringe comprising the solution. The syringe may befor example disposable and thus used once or manufactured for a multiuse routine.

In yet another aspect of the preset invention there is provided a kitwhich may comprise aqueous dispersion of surface-modified heavy metalnanoparticles dispersed in water or in an aqueous solution. In someembodiments, the aqueous solution comprises a buffer at pH values ofbetween 4.5 and 8; for example, at physiological pH values of about 7.3to 7.4, or pharmaceutical composition comprising the same means foradministering the aqueous dispersion or composition into a patient; andinstructions to use.

In another aspect of the present invention, it is provided a kit whichmay comprise surface-modified heavy metal nanoparticle in a dry form, anaqueous solution for dispersing the surface-modified heavy metalnanoparticle. In some embodiments, the aqueous dispersion compriseswater or a buffer at pH values of between 4.5 and 8, for example, atphysiological pH values of about 7.3 to 7.4, means for administering theaqueous dispersion into a patient; and instructions for use.

The aqueous solution dispersing the surface-modified heavy metalnanoparticle or the pharmaceutical composition comprising the same mayinclude additional components as long as the components are compatiblewith the dispersion, wherein compatible is to be understood as forexample components which do not cause precipitation or do not causeaggregation.

It should be appreciated that the advantageous stability of thesurface-modified heavy metal nanoparticles dispersions and compositionsprepared by the methods of preparation disclosed herein and used in thetreatment methods disclosed herein provide means and possibility tomaintain the surface-modified heavy metal nanoparticles in an aqueousdispersion for extended periods of time or alternatively, in a dry form.

Accordingly, the kits disclosed herein may comprise the aqueousdispersion of the heavy metal nanoparticles and means for administeringthe same, for example a sterile syringe. In an alternative embodimentthe sterile syringe may integrally comprise the pharmaceuticalcomposition comprising the aqueous dispersion of the heavy metalnanoparticles in a ready-to-use form.

The kit of the invention may comprise the surface-modified heavy metalnanoparticles in a dry form for example in a bottle, vial or ampoule,and an aqueous solution for dispersing them and thus in situ preparingan aqueous dispersions in either water or in a buffer solution at pHvalues of between 4.5 and 8; preferably at physiological pH values ofabout 7.3 to 7.4, means for injection of the composition such as asterile syringe.

The kit may comprise a sterile syringe which may comprisesurface-modified heavy metal nanoparticles in a dry form which uponusage are to be dispersed and thus preparing an aqueous dispersion ofthe nanoparticles in either water or in a buffer solution at pH valuesof, for example, between 4.5 and 8; for example, at physiological pHvalues of about 7.3 to 7.4, all within the syringe.

The invention further provides a method of treatment or diagnosis of amalignant disorder. The method of the invention comprises administeringto a subject in need a therapeutically effective amount ofsurface-modified heavy metal nanoparticles as described above or of apharmaceutically composition comprising the same. It should beappreciated that the method of the invention may optionally use the kitsas defined by the invention. The method of treatment or diagnosis maycomprise administrating by means of for example oral administration,infusion or injection.

Embodiments of the invention with gold as the heavy metal core of thenanoparticles are presented below, as non-limiting examples of thepractice of the invention, which can also employ other metal cores inthe nanoparticles compositions and dispersions according to theinvention.

As used in the specification and the appended claims and in accordancewith long-standing patent Law practice, the singular forms “a” “an” and“the” generally mean “at least one”, “one or more”, and other pluralreferences unless the context clearly dictates otherwise. Thus, forexample “a cell” and “a nanoparticle” include mixture of cells and oneor more nanoparticles of the type described.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

EXAMPLES

Material and Methods:

Oleic acid—65.0-88.0% (GC) (Aldrich)Ethanol—Frutarom (absolute—Chemically pure)HAuCl₄.3H₂O—ACS reagent, Sigma AldrichAscorbic acid—Cell culture tested, Sigma AldrichN-acetyl cysteine—Sigma grade, Sigma Aldrich.

Cysteine—≧97% (Aldrich), Sigma Aldrich NaOH—SigmaUltra, Sigma Aldrich

n-Hexane—Chemically pure, FrutaromEMT-6 cancer cells—mouse mammary carcinoma cell line [Technion, Israel]

DMEM, D-Glucose, FBS, L-Glutamine Solution, Pen-StrepSolution—Biological Industries

Trypsin-EDTA solution—Biological IndustriesAqua regia—Aqua Regia is a mixture of hydrochloric acid and nitric acidusually in a molar ratio of 3:1 respectively.

HCl—TraceSelect, Fluka Analytical HNO₃— TraceSelect, Fluka AnalyticalExample 1 Preparation of Gold Nanoparticles Coated with N-AcetylCysteine (NAC)

A mixture of fatty acids, (3.75 ml, 65% oleic acid, linoleic acid 18%,palmitic acid 16%, NaOH 200 mg, and ethanol (15 ml) were added to 30 mlwater (Milli-Q, Millipore) and the solution was allowed to stir for 5minutes on a magnetic stirrer.

Then, HAuCl₄ (50 mg) was added while stirring, resulting in a yellowishsolution, followed by 5 ml of 0.05M ascorbic acid which was slowly addedto the solution. When reduction of the gold species was completed (asindicated by the color of the solution turns to be red-wine), N-acetylcysteine (124 mg) was slowly added to the solution. The solution wasallowed to stir for 30 min.

The pH was then slowly adjusted to pH=9 using NaOH 2M and the resultingsolution was further allowed to stir for at least an hour on a magneticstirrer.

After that, n-hexane (20 ml) was added, and the solution continued to bestirred on a magnetic stirrer for at least an additional hour.

The pH of the resulting reaction mixture was then adjusted to pH=7 usingHCl solution (37%).

The mixture was separated into two phases, an organic phase and anaqueous phase, using a separation funnel. The aqueous phase wasevaporated under vacuum to remove traces of organic solvents. The driedpowder comprising surface-modified gold nanoparticles was dissolved in 5ml water (Milli-Q, Millipore), yielding an aqueous dispersion ofsurface-modified gold nanoparticles. The aqueous dispersion was dialyzedusing dialysis tube to remove unwanted products.

The aqueous dispersions of the surface-modified gold nanoparticlesprepared as described herein are present as separate nanoparticles asillustrated in FIGS. 1A and 1B. As shown in FIGS. 1A and 1B, thenanoparticles appearing as black “dots” are separated and do not formaggregates. In addition, as can be seen the gold nanoparticles arepresent at size equal to or small than 10 nm.

Example 2 Preparation of Gold Nanoparticles Coated with N-AcetylCysteine (NAC) and Albumin

Oleic acid (3.75 ml), NaOH 200 mg, and ethanol (15 ml) were added to 30ml of water (Milli-Q, Millipore), the solution was allowed to stir for 5minutes on a magnetic stirrer.

Then, HAuCl₄ (50 mg) was added to give a yellowish solution, whilestirring, 5 ml of 0.05 M ascorbic acid was added slowly to the solution.When reduction of the Au is completed (the color of the solution turnsto be red-wine) N-acetyl cysteine (111.6 mg in 5 ml water) was addedslowly to the solution. The solution was allowed to stir for 30 min.

The pH was then slowly adjusted to pH=9 using NaOH 2M and the resultingsolution was allowed to stir for at least one hour on a magneticstirrer. After that, n-hexane (20 ml) was added, the solution wascontinued to be stirred on a magnetic stirrer for at least oneadditional hour.

The pH of the resulting reaction mixture was adjusted to pH=7 using HClsolution (37%).

Then, excess albumin was added and the mixture was allowed to stir for30 min on the magnetic stirrer.

The mixture was separated into two phases, an organic phase and anaqueous phase using a separation funnel. The aqueous phase wasevaporated under vacuum to remove traces of organic solvents. The driedpowder comprising surface-modified gold nanoparticles was dissolved in 5ml water (Milli-Q, Millipore).

Example 3 Preparation of N-Acetyl Cysteine (NAC) and Polyethylene Glycol(PEG) Derivative

Materials and Methods:

Synthetic precursors (N-acetyl-L-cysteine >99%, and p-toluenesulfonicacid monohydrate >98.5%, and Poly (ethylene glycol)-600) were purchasedfrom Sigma-Aldrich without further purification. Solvents(Toluene >99.5%, methyl alcohol “analytical”, and dichloromethane“Ar”>99.9%) were purchased from Frutarom and Gadot, respectively, andused without further purification. Deuteriated solvents (DMSO-d6 andD2O) for NMR measurements were purchased from Sigma-Aldrich. Solventsfor chromatography were analytical grade. Sodium carbonate(Na2CO3)>99.8% and sodium sulfate (Na2SO4)>99% were purchased fromFrutarom. Analytical thin-layer chromatography (TLC) was performed onsilica-gel plates with F-254 indicator, visualized by irradiation withUV light. Column chromatography was carried out using silica-gel (Grace)0.040-0.063 mm (Merck). ¹H NMR spectra were recorded on a Bruker Avance400 (400 MHz ¹H NMR). Chemical shift values (δ) are reported in ppm (TMSδ=0 ppm for 1H; residual DMSO-d6 δ=2.5 ppm for 1H). The proton spectraare reported as follows δ (multiplicity, coupling constant J, number ofprotons, moiety). Multiplicities are indicated by s (singlet), t(triplet), m (multiplet), and so on.

Preparation of NAC:PEG Molecule:

A 250 mL, one-necked, round-bottomed flask equipped with a magneticstirrer, Dean-Stark trap, and a reflux condenser. The flask was chargedwith N-acetylcysteine, (“NAC”, 3.26 g, 20 mmol) and poly(ethyleneglycol)-600, (“PEG-600”, 12.0 g, 20 mmol)) in toulene (150 mL).p-toluenesulfonic acid monohydrate (“p-TSA”, 4.0 g, 21 mmol) was addedand the stirred mixture was heated under reflux in an oil bath (about140° C.) for 2-3 hr (the reaction was monitored by TLC). The mixture wasallowed to cool to ambient temperature and two phases were observed. Themixture was neutralized by adding sodium carbonate (2 g) and stirred for2 hrs (gas was bubbled). The solvent was evaporated under reducedpressure and the residue was dissolved in dichloromethane, theprecipitate was filtered, the organic phase was dried over dry sodiumsulfate, filtered and concentrated with a rotary evaporator to give aviscous oil.

The product was purified by column chromatography on silica gel,(eluent: CH2Cl2:CH3OH, 95:5). The appropriate fractions were collectedand concentrated by a rotary evaporator to give 7.4 g (9.7 mmol, 48.5%)of the product as a yellow viscous oil. ¹H-NMR (DMSO-d6, 400 MHz) δ:1.82 (s, 1H, SH), 2.13 (s, 3H, CH3), 3.34-3.60 (m, 56H, PEG-CH2O),4.13-4.22 (m, 2H, CH2S), J=8.1 Hz, 1H, CH), 5.69 (s, 1H, NH).

The ¹H-NMR Spectrum of NAC-PEG is shown in FIG. 2.

To prepare NAC-PEG coated nanoparticles, 10 mg/ml gold nanoparticleswere incubated for 24 hr at 25° C. with PEG-NAC solution (prepared asdescribed above) at a molar ratio of 1:1.

Example 4 Preparation of Gold Nanoparticles Coated with Cysteine

Oleic acid (3.75 ml), NaOH 200 mg, and ethanol (15 ml) are added to 30ml water

(Milli-Q, Millipore) and the solution is allowed to stir for 5 minuteson a magnetic stirrer.

Then, HAuCl₄ (50 mg) is added to give a yellowish solution, whilestirring, 5 ml of 0.05 M ascorbic acid is added slowly to the solution.When reduction of the Au is completed (the color of the solution turnsto be red-wine) cysteine (92.6 mg in 5 ml water) is added slowly to thesolution. The solution is allowed to stir for 30 min.

The pH is then slowly adjusted to pH=9 using NaOH 2M and the resultingsolution is allowed to stir for at least one hour on a magnetic stirrer.After that, n-hexane (20 ml) is added, the solution is continued to bestirred on a magnetic stirrer for at least one additional hour.

The pH of the resulting reaction mixture is adjusted to pH=7 using HClsolution (37%).

The mixture is separated into two phases, an organic phase and anaqueous phase using a separation funnel. The aqueous phase is evaporatedunder vacuum to remove traces of organic solvents. The dried powdercomprising surface-modified gold nanoparticles is dissolved in 5 mlwater (Milli-Q, Millipore).

Example 5 Gold Nanoparticles with a Coat Contains 90% Cysteine and 10%Albumin

Oleic acid (3.75 ml), NaOH 200 mg, and ethanol (15 ml) are added to 30ml water (Milli-Q, Millipore), the solution is allowed to stir for 5minutes on a magnetic stirrer.

Then, HAuCl₄ (50 mg) is added to give a yellowish solution, whilestirring, 5 ml of 0.05 M ascorbic acid is added slowly to the solution.When reduction of the Au is completed (the color of the solution turnsto be red-wine) cysteine (83.34 mg in 5 ml water) is added slowly to thesolution. The solution is allowed to stir for 30 min.

The pH is then slowly adjusted to pH=9 using NaOH 2M and the resultingsolution is allowed to stir for at least one hour on a magnetic stirrer.After that, n-hexane (20 ml) is added, the solution is continued to bestirred on a magnetic stirrer for at least one additional hour.

The pH of the resulting reaction mixture is adjusted to pH=7 using HClsolution (37%).

Then, albumin is added and the mixture is allowed to stir for 30 min onthe magnetic stirrer.

The mixture is separated into two phases, an organic phase and anaqueous phase using a separation funnel. The aqueous phase is evaporatedunder vacuum to remove traces of organic solvents. The dried powdercomprising surface-modified gold nanoparticles is dissolved in 5 mlwater (Milli-Q, Millipore).

Example 6 Stability of NAC-Surface-Modified Gold Nanoparticles or ofPEG-NAC Surface Modified Gold Nanoparticles in Protein ContainingSolutions

The stability of NAC-surface-modified gold nanoparticles orPEG-NAC-surface-modified gold nanoparticles was tested in growth medium(DMEM). Culture plates were incubated with 9.5 ml of DMEM and 500 μl ofNAC-surface-modified gold nanoparticles or PEG-NAC surface-modified goldnanoparticles for 24 hours.

The results are presented in FIG. 4, which shows pictograms of cellculture medium dish containing PEG-NAC surface coated gold nanoparticles(left hand dish) or cell culture medium dish containing NAC surfacecoated gold nanoparticles (right hand dish). As shown in FIG. 4, thecell culture medium dish containing PEG-NAC-surface-modified goldnanoparticles appears clear (left hand dish) as compared to cell culturemedium contains the same concentration of NAC surface coated goldnanoparticle, in which formation of GNPs-protein conjugates is observed(right hand dish). Thus, the PEG-NAC surface modification providesenhanced solubility and stability to the gold nanoparticles as comparedto surface modification by NAC alone.

Likewise, the stability of gold nanoparticles or PEG-NACsurface-modified gold nanoparticles was tested in the following mediums:Blood, growth medium (DMEM) and fetal bovine serum (FBS) solution.

Cultures plates containing 9.5 ml of DMEM or FBS were incubated with 500μl of gold nanoparticles or PEG-NAC surface-modified gold nanoparticlesand tracked for 24 hours. tubes containing 2.5 ml of blood wereincubated with 300 μl of gold nanoparticles or PEG-NAC surface-modifiedgold nanoparticles.

The results show that plates that were incubated with goldnanoparticles, aggregates were formed and black deposits wereindentified, which are indicators of unstable nanoparticles. Incontrast, plates that were incubated with PEG-NAC surface-modified goldnanoparticles, showed clear solution, with no formation of aggregates orother deposits, indicating that the surface-modified gold nanoparticlesare stable in such solutions.

The results presented in FIGS. 5A-B are of light-microscope images ofblood samples incubated with 300 μl of gold nanoparticles or withNAC-PEG surface-modified gold nanoparticles. The results shown in FIGS.5A-5B demonstrate that whereas in blood incubated with 300 μl of goldnanoparticles, aggregates were formed (FIG. 5A), in blood incubated with300 μl of PEG-NAC surface-modified gold nanoparticles, no aggregationwas detected and the blood solution remained clear (FIG. 5B). [

Example 7 In Vitro Assay—Introduction of Gold Nanoparticles to CancerCells

EMT-6 cancer cells were grown in Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% FBS, 2% L-Glutamine Solution (200 mM) and 2%Pen-Strep Solution (Biological Industries) and 4.5 m/1 D-Glucose underregular growth conditions: 5% CO₂, and 37.6° C.

An aqueous dispersion of surface-modified gold nanoparticles preparedaccording to the procedure of Example 1, was added to the cells at oneof the final concentrations of 0.02 mg/ml, 0.04 mg/ml. 0.127 mg/ml or0.253 mg/ml for an incubation periods of 2 hours or 4 hours in theincubator, control cells were kept in the same conditions albeit theaddition of gold nanoparticles.

After the incubation times of 2 h or 4 h, cells were washed with PBS,harvested using Trypsin-EDTA solution (Biological Industries), countedunder microscope using a microscope counting chamber (Hemocytometer) anddigested with aqua regia for ICP (Inductivity Coupled PlasmaSpectroscopy) analysis where the threshold value is set to 1 ppb.

Gold concentration in each sample was determined using the Varian 720-ESICP instrument.

The amount of Au in 1 g cells was calculated. Control groups were alsoanalyzed for gold, the results demonstrated the absence of gold in thecontrol samples.

Determination of gold concentrations following incubation of the cellwith aqueous dispersions of surface-modified gold nanoparticles was doneusing the Varian 720-ES (Inductively coupled plasma mass spectrometry)ICP-MS instrument. ICP-MS is a mass spectrometry instrument that ishighly sensitive and capable of determining metal concentration.

As can be seen in Tables 1 and 2, the aqueous dispersions ofsurface-modified gold nanoparticles are incorporated into the cells atthe concentrations and time period tested.

As can be seen in Table 1, incorporation of gold nanoparticles to thecancer cells is dependent on the concentration of the gold nanoparticlesin the growth medium, and on the incubation times. There was no toxicityobserved in the cells during the incubation of the gold nanoparticles asindicated by comparing the number of the treated cells to the controlcells.

In a further study, higher concentrations of gold nanoparticles wereused, as can be seen in Table 2, the dependency on the goldnanoparticles concentration and time of incubation is maintained.

More importantly, even at higher concentration no toxic effects on thecells were observed. Therefore, suggesting the safe use of the aqueousdispersions of surface-modified gold nanoparticles in in vivo studies,

TABLE 1 Gold concentrations in cells. Gold concentration in cells medium0.02 mg/ml 0.04 mg/ml Calculated Calculated Incubation time Au(mg)/g incells Au(mg)/g in cells 2 hours-1 1.53 2.68 2 hours-2 1.72 3.05 4hours-1 2.70 4.66 4 hours-2 1.17 4.00

TABLE 2 Gold concentrations in cells. Gold concentration in cells medium0.127 mg/ml 0.253 mg/ml Calculated Calculated Incubation time Au(mg)/gin cells Au(mg)/g in cells 5.5 5.19 7.12 0.5 hour-2 5.02 5.41 1 hour-15.99 13.2 1 hour-2 5.39 17.8 3 hour-1 18 32.9 3 hour-2 16.4 33.2

Example 8 Delivery of Gold Nanoparticles to Tumors and Various Tissues

Balb/C mice (3 groups of 15 mice each) bearing tumors in their thighs(KHJJ line, murine mammary carcinoma) are used in these experiments toshow the delivery of gold nanoparticles into tumors and various tissues.

The first experimental group is a control group of mice with an inducedtumor, that are injected intravenously (i.v.) with physiologicalsolution (saline). The mice in the second experimental group areinjected i.v. with 50 mg/kg of an aqueous dispersion of surface-modifiedgold nanoparticles prepared according to examples 1 to 4. The mice inthe third experimental group are injected i.v. with 50 mg/kg of anaqueous dispersion of surface-modified gold nanoparticles, modified withN-acetyl cysteine and PEG-cysteine.

Tissue samples (kidneys, liver, spleen, lungs, heart and brain) from thedifferent groups are analyzed for the presence of gold using the ICPinstrument at 4 hours, 8 hours, 24 hours, 48 hours and 72 hourspost-injection.

Example 9 Determination of the PEG-NAC Surface Modified GoldNanoparticles Maximum Tolerated Dose (MTD)

Mice are repeatedly injected (qd) with increasing doses of aqueousdispersions of PEG-NAC surface-modified gold nanoparticles and areobserved for general toxicity signs for a time period of 12-14 days:

I. MTD Assessment Following Multi Dose (IV) Injection

Determination of the MTD (Maximum Tolerated Dose) of gold nanoparticles(GNPs) was assessed following multi dose intravenous (IV) injection to 5female nude mice, age 8-12 weeks, body weight 18-22 g. Daily dose of100, 150 and 200 mg/kg, treatment schedule as detailed below. Dosingvolume of 10 mL/kg (0.2 mL/20 g mouse). The endpoint of the study wasgroup mean weight loss exceeds 20% or death of >10% of animals in thegroup. Study Endpoint of 26 days.

The experimental groups were as follows:

-   Group 1-5 female nude mice injected (i.v.) with 100 mg/kg gold    nanoparticles every other day for six days (qod×6).-   Group 2-5 female nude mice injected (i.v.) with 100 mg/kg gold    nanoparticles for 5 days, two days off and 5 more days (5/2/5).-   Group 3-5 female nude mice injected (i.v.) with 150 mg/kg gold    nanoparticles for 5 days, two days off and 5 more days (5/2/5).-   Group 4-5 female nude mice injected (i.v.) with 200 mg/kg gold    nanoparticles for 5 days, two days off and 5 more days (5/2/5).

Results:

The results are presented in FIGS. 3A-D, which shows graphs of change inmean body weight (g) over time (days after injection) of the varioustreated groups. FIG. 3A—change in body weight of group I mice (treatedwith 100 mg/kg gold nanoparticles every other day, for six days). FIG.3B-change in body weight of group II mice (treated with 100 mg/kg goldnanoparticles, iv, for five days, two days off and 5 more days (5/2/5)).FIG. 3C—change in body weight of group III mice (treated with 150 mg/kggold nanoparticles, iv, 5/2/5). FIG. 3D-change in body weight of groupIV mice (treated with 200 mg/kg gold nanoparticles, iv, 5/2/5). Nomortality occurred in the animals treated with the GNPs prior to thescheduled termination, carried out 26 days post-dosing.

The results demonstrate that no statistically significance loss in meanbody weight was confined to any of the GNPs treated groups.

II. MTD Assessment Following Multi Dose (IV) Injection in Combinationwith Radiation

Determination of the MTD (Maximum Tolerated Dose) of Gold nano Particleswas assessed following multi dose intravenous (IV) injection to 5 femalenude mice (age 8-12 weeks, body weight 18-22 g) in combination withexternal irradiation.

Daily Dose of 100, 150 and 200 mg/kg combined with radiation daily doseof 2 Gy, treatment schedule of 5/2/5. Dosing volume of 10 mL/kg (0.2mL/20 g mouse). The endpoint of the study was group mean weight lossexceeds 20% or death of >10% of animals in the group. Study Endpoint of26 days

Results:

The results are presented in FIG. 3E, which shows graphs of percentchange in body weight (g) over time (days after injection) of grouptested with 200 mg/kg of PEG-NAC surface modified gold nanparticles,alone (G1) or in combination with irradiation (G2).

No mortality occurred in the animals treated with the GNPs prior to thescheduled termination, carried out 26 days post-dosing.

No statistically significance loss in mean body weight was confined toany of the GNPs treated groups.

The results indicate that doses of 200 mg/kg and below are safe doses.

Example 10 Toxicity Study

Mice are treated with aqueous dispersions of surface-modified goldnanoparticles and CBC and blood chemistry are determined.

Blood chemistry parameters include: ALKP, amylase, bilirubin, BUN,calcium, cholesterol, ALT, phosphorus ALB, creatine, protein, NH₃, AST,Gk, GCT, Glucose, LDH, lipase, magnesium, triglycerides and uric acid.

CBC analysis: Analysis via Forcyte CBC analyzer, samples is measured nomore than 4 hours after extraction. Parameters include: measurement ofhematocrit (HCT), hemoglobin (HGB), Mean Corpuscular HemoglobinConcentration (MCHC), Mean Corpuscular Hemoglobin (MCH), MeanCorpuscular Volume (MCV), platelets, Red Blood Cell Count (RBC), andWhite Blood Cell Count (WBC).

Example 11 Pharmacokinetics (PK) Studies

Collecting tumor and blood samples from animals bearing H460 human Nonsmall cell lung carcinoma (NSCLC), treated with 200 mg/Kg non-surfacemodified gold nanoparticles or 200 mg/Kg of PEG-NAC surface modifiedgold nanoparticles.

CR female NCr nu/nu mice, age 8 to 12 weeks were injected with 1×10⁷H460 tumor cells sc in flank. Cell injection volume is 0.2 mL/mouse.When tumors reach an average size of 250-350 mg, pair match were doneand treatment started. PEG-NAC surface modified gold nanoparticles wereinjected to group 1 mice and PEG-NAC composition was injected to thesecond group at a dosing volume of 10 mL/kg (0.200 mL/20 g mouse).Volume was adjusted according to the body weight. Gold nanoparticlesdose was 200 mg/Kg.

The results are presented in FIGS. 6A-D. FIG. 6A is a graph showing thepharmacokinetics of NAC surface-modified gold nanoparticles in plasma orin tumors of mice bearing tumors. The graph shows the concentration ofgold in plasma or in tumor of mice that were injected with the NACsurface-modified gold nanoparticles (hours after injection). FIG. 6B isa graph showing the pharmacokinetics of PEG-NAC surface-modified goldnanoparticles in plasma or in tumors of mice bearing tumors. The graphshows the concentration of gold in plasma or in tumor of mice bearingtumors that were injected with the PEG-NAC surface-modified goldnanoparticles (hours after injection).

FIGS. 6C-D, shows the percent injected dose per gram tumor or per gramplasma, respectively. FIG. 6C is a graph showing the percent injecteddose (%) per gram tumor over time (days after injection) in tumor, ofPEG-NAC surface modified gold nanoparticles (tumor AuPEG-GNPs) or ofNAC-surface modified gold nanoparticles (“regular GNPs”). FIG. 6D is agraph showing the percent injected dose (%) per gram over time (daysafter injection) in plasma, of PEG-NAC surface modified goldnanoparticles (AuPEG-GNPs or of NAC-surface modified gold nanoparticles(“regular GNPs”).

The results indicate that higher tumor levels were obtained from thePEG-NAC surface modified gold nanoparticles. For PEG-NAC surfacemodified gold nanoparticles, gold level maximize at 24-72 hours post theinjection of the nanoparticles.

Long circulation time (as determined by half-life at blood) was obtainedfor the PEG-GNPs.

All together, the results demonstrate that the novel surfacemodification with PEG-NAC improves pharmacokinetics of goldnanoparticles.

1. A surface-modified heavy metal nanoparticle comprising: a heavy metalcore and a coating layer, the coating layer comprising at least oneligand conjugated to polyethylene glycol (PEG), wherein the at least oneligand is selected from N-acetyl cysteine (NAC), albumin, cysteine,methionine, glutathione, amino thiols, thio-carboxylic acids, ammonia,amines, diamines or any combination thereof.
 2. The surface-modifiedheavy metal nanoparticle of claim 1 wherein the ligand is N-acetylcysteine (NAC).
 3. The surface-modified heavy metal nanoparticle ofclaim 1, wherein the heavy metal is selected from gold, gold species,silver, platinum, iron, copper, nickel, palladium, iridium, titanium orlead.
 4. The surface-modified heavy metal nanoparticle of claim 1,wherein the heavy metal is a gold species.
 5. An aqueous dispersion ofsurface-modified heavy metal nanoparticles of claim 1, dispersed inwater or in a buffer.
 6. The aqueous dispersion of claim 5, wherein thesurface-modified heavy metal nanoparticles are stable at physiologicalconditions.
 7. The aqueous dispersion of claim 5, wherein thesurface-modified heavy metal nanoparticles are present at concentrationsof 0.001 μM-1M.
 8. A process for the preparation of surface-modifiedheavy metal nanoparticles comprising the steps of: adding at least oneligand conjugated to poly ethylene glycol (PEG) to a mixture comprisingmetal nanoparticles, wherein the at least one ligand binds to thesurface of the heavy metal nanoparticles core, yielding surface-modifiedheavy metal nanoparticle, wherein the ligand is selected from N-acetylcysteine (NAC), albumin, cysteine, methionine, glutathione, aminothiols, thio-carboxylic acids, ammonia, amines, diamines or anycombination thereof.
 9. The process of claim 8, wherein the ligand isN-acetyl cysteine (NAC).
 10. The process of claim 8, wherein the mixtureis prepared by: (a) mixing at least one surfactant, comprising at leastone fatty acid, with at least one organic solvent in a water solution toyield an emulsion; (b) adding to the emulsion of step (a) a solution ofheavy metal species and at least one reducing agent, to yield reducedmetal nanoparticles.
 11. The process claim 8, further comprisingseparating the inorganic phase which contains the surface-modified heavymetal nanoparticles from the organic phase.
 12. The process of claim 8,wherein the heavy metal is a gold species.
 13. The process of claim 12,wherein the gold species is obtained from AuCl₃, AuF₃, AuBr₃, HAuCl₄ orMAuCl₄, wherein M represents an alkali metal cation.
 14. The process ofclaim 10, further comprising adding at least one second organic solvent,selected from the group consisting of hexane, cyclohexane, chloroform,diethyl ether, ethyl acetate and toluene.
 15. A surface-modified heavymetal nanoparticle or an aqueous dispersion of surface-modified heavymetal nanoparticles, obtainable by the process according to claim
 8. 16.A pharmaceutical or a diagnostic composition comprising surface-modifiedheavy metal nanoparticles according to claim
 1. 17. A kit comprising:(a) aqueous dispersion of the surface-modified heavy metal nanoparticlesof claim 1, wherein the dispersion comprises water or a buffer at pHvalues of between about 4.5 to about 8 or pharmaceutical compositioncomprising the same, or the surface-modified heavy metal nanoparticlesof claim 1, in a dry form and an aqueous solution for dispersing thesurface-modified heavy metal nanoparticles, wherein the aqueousdispersion comprises water or a buffer at pH values of between 4.5 and8; (b) means for administering the aqueous dispersion or pharmaceuticalcomposition into a patient; and (c) instructions for use.
 18. A methodof treatment or diagnosis of a malignant disorder or a cellproliferative disorder comprising the steps of administering a subjectin need a therapeutically effective amount of surface-modified heavymetal nanoparticles as defined in claim 1 or of a pharmaceuticallycomposition comprising the same.
 19. The method of claim 18, wherein themalignant disorder is selected from the group consisting of carcinoma,sarcoma, germ cell tumors and blastoma.
 20. An N-acetyl cysteine(NAC)-polyethylene glycol (PEG) conjugate for use in surfacemodification of metal nanoparticles, wherein the N-acetyl cysteine (NAC)is capable of bounding to the surface of the metal nanoparticle core.